Human sel-10 polypeptides and polynucleotides that encode them

ABSTRACT

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding either of two alternative splice variants of human sel-10, one of which is expressed in hippocampal cells, and one of which is expressed in mammary cells. The invention also provides isolated sel-10 polypeptides and cell lines which express them in which Aβ processing is altered.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/213,888, filed Dec.17, 1998, which claims the benefit of the following provisionalapplication: U.S. Ser. No. 60/068,243, filed 19 Dec. 1997, under 35 USC119(e)(1).

FIELD OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding either of two alternative splicevariants of human sel-10, one of which is expressed in hippocampalcells, and one of which is expressed in mammary cells. The inventionalso provides isolated sel-10 polypeptides.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a degenerative disorder of the centralnervous system which causes progressive memory and cognitive declineduring mid to late adult life. The disease is accompanied by a widerange of neuropathologic features including extracellular amyloidplaques and intra-neuronal neurofibrillary tangles. (Sherrington, R., etal.; Nature 375: 754-60 (1995)). Although the pathogenic pathway leadingto AD is not well understood, several genetic loci are known to beinvolved in the development of the disease.

Genes associated with early onset Alzheimer's disease (AD) have beenidentified by the use of mapping studies in families with early-onsetAD. These studies have shown that genetic loci on chromosomes 1 and 14were likely to be involved in AD. Positional cloning of the chromosome14 locus identified a novel mutant gene encoding an eight-transmembranedomain protein which subsequently was named presenilin-1 (PS-1).(Sherrington, R., et al.; Nature 375: 754-60 (1995)). Blast search ofthe human EST database revealed a single EST exhibiting homology toPS-1, designated presenilin-2 (PS-2) which was shown to be the geneassociated with AD on chromosome 1. (Levy-Lahad, E. et al., Science269:973-977 (1995); Rogaev, E. I., et al., Nature 376: 775-8 (1995); Li,J. et al., Proc. Natl. Acad. Sci. U.S.A. 92: 12180-12184 (1995)).altered by the point mutations found in familial Alzheimer's disease[Perez-Tur, J. et al., Neuroreport 7: 297-301 (1995); Mercken, M. etal., FEBS Lett. 389: 297-303 (1996)]. PS-1 gene expression is widelydistributed across tissues, while the highest levels of PS-2 mRNA arefound in pancreas and skeletal muscle. (Li, J. et al., Proc. Natl. Acad.Sci. U.S.A. 92: 12180-12184 (1995); Jinhe Li, personal communication).The highest levels of PS-2 protein, however, are found in brain (JinheLi, personal communication). Both PS-1 and PS-2 proteins have beenlocalized to the endoplasmic reticulum, the Golgi apparatus, and thenuclear envelope. (Jinhe Li, personal communication; Kovacs, D. M. etal., Nat. Med. 2:224-229 (1996); Doan, A. et al., Neuron 17: 1023-1030(1996)). Mutations in either the PS-1 gene or the PS-2 gene alter theprocessing of the amyloid protein precursor (APP) such that the ratio ofA-beta₁₋₄₂ is increased relative to A-beta₁ ₄₀ (Scheuner, D. et al.,Nat. Med. 2: 864-870 (1996)). When coexpressed in transgenic mice withhuman APP, a similar increase in the ratio of A-beta₁₋₄₂ as compared toA-beta₁₋₄₀ is observed (Borchelt, D. R. et al., Neuron 17: 1005-1013(1996); Citron, M. et al., Nat. Med. 3: 67-72 (1997); Duff, K. et al.,Nature 383: 710-713 (1996)), together with an acceleration of thedeposition of A-beta in amyloid plaques (Borchelt et al., Neuron 19: 939(1997).

Despite the above-described observations made with respect to the roleof PS-1 and PS-2 in AD, their biological function remains unknown,placing them alongside a large number of human disease genes having anunknown biological function. Where the function of a gene or its productis unknown, genetic analysis in model organisms can be useful in placingsuch genes in known biochemical or genetic pathways. This is done byscreening for extragenic mutations that either suppress or enhance theeffect of mutations in the gene under analysis. For example, extragenicsuppressors of loss-of-function mutations in a disease gene may turn onthe affected genetic or biochemical pathway downstream of the mutantgene, while suppressers of gain-of-function mutations will probably turnthe pathway off.

One model organism that can be used in the elucidation of the functionof the presenilin genes is C. elegans, which contains three genes havinghomology to PS-1 and PS-2, with sel-12 having the highest degree ofhomology to the genes encoding the human presenilins. Sel-12 wasdiscovered in a screen for genetic suppressers of an activated notchreceptor, lin-12(d) (Levitan, D. et al., Nature 377: 351-354 (1995)).Lin-12 functions in development to pattern cell lineages. Hypermorphicmutations such as lin-12(d), which increase lin-12 activity, cause a“multi-vulval” phenotype, while hypomorphic mutations which decreaseactivity cause eversion of the vulva, as well as homeotic changes inseveral other cell lineages (Greenwald, I., et al., Nature 346: 197-199(1990); Sundaram, M. et al., Genetics 135: 755-763 (1993)). Sel-12mutations suppress hypermorphic lin-12(d) mutations, but only if thelin-12(d) mutations activate signaling by the intact lin-12(d) receptor(Levitan, D. et al., Nature 377: 351-354 (1995)). Lin-12 mutations thattruncate the cytoplasmic domain of the receptor also activate signaling(Greenwald, I., et al., Nature 346: 197-199 (1990)), but are notsuppressed by mutations of sel-12 (Levitan, D. et al., Nature 377:351-354 (1995)). This implies that sel-12 mutations act upstream of thelin-12 signaling pathway, perhaps by decreasing the amount of functionallin-12 receptor present in the plasma membrane. In addition tosuppressing certain lin-12 hypermorphic mutations, mutations to sel-12cause a loss-of-function for egg laying, and thus internal accumulationof eggs, although the mutants otherwise appear anatomically normal(Levitan, D. et al., Nature 377: 351-354 (1995)). Sel-12 mutants can berescued by either human PS-1 or PS-2, indicating that sel-12, PS-1 andPS-2 are functional homologues (Levitan, D., et al., Proc. Natl. Acad.Sci. U.S.A 93: 14940-14944 (1996)).

A second gene, sel-10, has been identified in a separate genetic screenfor suppressors of lin-12 hypomorphic mutations. Loss-of-functionmutations in sel-10 restore signaling by lin-12 hypomorphic mutants. Asthe lowering of sel-10 activity elevates lin-12 activity, it can beconcluded that sel-10 acts as a negative regulator of lin-12 signaling.Sel-10 also acts as a negative regulator of sel-12, the C. eleganspresenilin homologue (Levy-Lahad, E. et al., Science 269:973-977(1995)). Loss of sel-10 activity suppresses the egg laying defectassociated with hypomorphic mutations in sel-12 (Iva Greenwald, personalcommunication). The effect of loss-of-function mutations to sel-10 onlin-12 and sel-112 mutations indicates that sel-10 acts as a negativeregulator of both lin-12/notch and presenilin activity. Thus, a humanhomologue of C. elegans sel-10 would be expected to interact geneticallyand/or physiologically with human presenilin genes in ways relevant tothe pathogenesis of Alzheimer's Disease.

In view of the foregoing, it will be clear that there is a continuingneed for the identification of genes related to AD, and for thedevelopment of assays for the identification of agents capable ofinterfering with the biological pathways that lead to AD.

Information Disclosure

-   Hubbard E J A, Wu G, Kitajewski J, and Greenwald I (1997) Sel-10, a    negative regulator of lin-12 activity in Caenorhabditis elegans,    encodes a member of the CDC4 family of proteins. Genes & Dev    11:3182-3193.-   Greenwald-I; Seydoux-G (1990) Analysis of gain-of-function mutations    of the lin-12 gene of Caenorhabditis elegans. Nature. 346: 197-9-   Kim T-W, Pettingell W H, Hallmark O G, Moir R D, Wasco W, Tanzi    R (1997) Endoproteolytic cleavage and proteasomal degradation of    presenilin 2 in transfected cells. J Biol Chem 272:11006-11010.-   Levitan-D; Greenwald-I (1995) Facilitation of lin-12-mediated    signalling by sel-12, a Caenorhabditis elegans S182 Alzheimer's    disease gene. Nature. 377: 351-4.-   Levitan-D; Doyle-T G; Brousseau-D; Lee-M K; Thinakaran-G; Slunt-H H;    Sisodia-S S; Greenwald-I (1996) Assessment of normal and mutant    human presenilin function in Caenorhabditis elegans. Proc. Natl.    Acad. Sci. U.S.A. 93: 14940-4.-   Sundaram-M; Greenwald-I (1993) Suppressors of a lin-12 hypomorph    define genes that interact with both lin-12 and glp-1 in    Caenorhabditis elegans. Genetics. 135: 765-83.-   Sundaram-M; Greenwald-I (1993) Genetic and phenotypic studies of    hypomorphic lin-12 mutants in Caenorhabditis elegans. Genetics. 135:    755-63.-   F55B12.3 GenPep Report (WMBL locus CEF55B12, accession z79757).-   WO 97/11956

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding human sel-10, which is expressed inhippocampal cells and in mammary cells. Unless otherwise noted, anyreference herein to sel-10 will be understood to refer to human sel-10,and to encompass both hippocampal and mammary sel-10. Fragments ofhippocampal sel-10 and mammary sel-10 are also provided.

In a preferred embodiment, the invention provides an isolated nucleicacid molecule comprising a polynucleotide having a sequence at least 95%identical to a sequence selected from the group consisting of:

-   -   (a) a nucleotide sequence encoding a human sel-10 polypeptide        having the complete amino acid sequence selected from the group        consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID        NO:6, and SEQ ID NO:7, or as encoded by the cDNA clone contained        in ATCC Deposit No.98978;    -   (b) a nucleotide sequence encoding a human sel-10 polypeptide        having the complete amino acid sequence selected from the group        consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or as        encoded by the cDNA clone contained in ATCC Deposit No. 98979;        and    -   (c) a nucleotide sequence complementary to the nucleotide        sequence of (a) or (b).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringentconditions to a polynucleotide encoding sel-10, or fragments thereof.

The present invention also provides vectors comprising the isolatednucleic acid molecules of the invention, host cells into which suchvectors have been introduced, and recombinant methods of obtaining asel-10 polypeptide comprising culturing the above-described host celland isolating the sel-10 polypeptide.

In another aspect, the invention provides isolated sel-10 polypeptides,as well as fragments thereof. In a preferred embodiment, the sel-10polypeptides have an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, and 10. Isolatedantibodies, both polyclonal and monoclonal, that bind specifically tosel-10 polypeptides are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: FIGS. 1A and 1B are western blots showing proteinexpression in HEK293 cells transfected with PS1—C-FLAG, 6-myc-N-sel-10,and APP695NL-KK cDNAs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding human sel-10. The nucleotidesequence of human hippocampal sel-10 (hhsel-10), which sequence is givenin SEQ ID NO:1, encodes five hhsel-10 polypeptides (hhsel-10-(1),hhsel-10-(2), hhsel-10-(3), hhsel-10-(4), and hhsel-10-(5), referred tocollectively herein as hhsel-10). The nucleotide sequence of humanmammary sel-10 (hmsel-10), which sequence is given in SEQ ID NO:2,encodes three hmsel-10 polypeptides (hmSel-10-(1), hmSel-10-(2), andhmsel-10-(3), referred to collectively herein as hmsel-10). Thenucleotide sequences of the hhsel-10 polynucleotides are given in SEQ IDNO. 1, where nucleotide residues 45-1928 of SEQ ID NO. 1 correspond tohhsel-10-(1), nucleotide residues 150-1928 of SEQ ID NO. 1 correspond tohhSel-10-(2), nucleotide residues 267-1928 of SEQ ID NO. 1 correspond tohhSel-10-(3), nucleotide residues 291-1928 of SEQ ID NO. 1 correspond tohhSel-10-(4), and nucleotide residues 306-1928 of SEQ ID NO. 1correspond to hhSel-10-(5). The nucleotide sequences of the hmSel-10polynucleotides are given in SEQ ID NO. 2, where nucleotide residues180-1949 of SEQ ID NO. 2 correspond to hmSel-10-(1), nucleotide residues270-1949 of SEQ ID NO. 2 correspond to hmSel-10-(2), and nucleotideresidues 327-1949 of SEQ ID NO. 2 correspond to hmSel-10-(3). The aminoacid sequences of the polypeptides encoded by the hhSel-10 and hm-Sel-10nucleic acid molecules are given as follows: SEQ ID NOS: 3, 4, 5, 6, and7 correspond to the hhSel-10-(1), hhSel-10-(2), hhSel-10-(3),hhSel-10-(4), and hhSel-10-(5) polypeptides, respectively, and SEQ IDNOS: 8, 9, and 10 correspond to the hmSel-10-(1), hmSel-10-(2), andhmSel-10-(3) polypeptides, respectively. Unless otherwise noted, anyreference herein to sel-10 will be understood to refer to human sel-10,and to encompass all of the hippocampal and mammary sel-10 nucleic acidmolecules (in the case of reference to sel-10 nucleic acid,polynucleotide, DNA, RNA, or gene) or polypeptides (in the case ofreference to sel-10 protein, polypeptide, amino acid sequnce). Fragmentsof hippocampal sel-10 and mammary sel-10 nucleic acid molecules andpolypeptides are also provided.

The nucleotide sequence of SEQ ID NO:1 was obtained as described inExample 1, and is contained in cDNA clone PNV 102-1, which was depositedon Nov. 9, 1998, at the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110, and given accession number 98978.The nucleotide sequence of SEQ ID NO:2 was obtained as described inExample 1, and is contained in cDNA clone PNV 108-2, which was depositedon Nov. 9, 1998, at the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110, and given accession number 98979.

The human sel-10 polypeptides of the invention share homology with C.elegans sel-10, as well as with members of the β-transducin proteinfamily, including yeast CDC4, and human LIS-1. This family ischaracterized by the presence of an F-box and multiple WD-40 repeats(Li, J., et al., Proc. Natl. Acad. Sci. U.S.A. 92:12180-12184 (1995)).The repeats are 20-40 amino acids long and are bounded by gly-his (GH)and trp-asp (WD) residues. The three dimensional structure ofβ-transducin indicates that the WD40 repeats form the arms of aseven-bladed propeller like structure (Sondek, J., et al., Nature379:369-374 (1996)). Each blade is formed by four alternating pleats ofbeta-sheet with a pair of the conserved aspartic acid residues in theprotein motif forming the limits of one internal beta strand. WD40repeats are found in over 27 different proteins which represent diversefunctional classes (Neer, E. J., et al., Nature 371:297-300 (1994)).These regulate cellular functions including cell division, cell fatedetermination, gene transcription, signal transduction, proteindegradation, mRNA modification and vesicle fusion. This diversity infunction has led to the hypothesis that β-transducin family membersprovide a common scaffolding upon which multiprotein complexes can beassembled.

The nucleotide sequence given in SEQ ID NO:1 corresponds to thenucleotide sequence encoding hhsel-10, while the nucleotide sequencegiven in SEQ ID NO:2 corresponds to the nucleotide sequence encodinghmsel-10. The isolation and sequencing of DNA encoding sel-10 isdescribed below in Examples 1 and 2.

As is described in Examples 1 and 2, automated sequencing methods wereused to obtain the nucleotide sequence of sel-10. The sel-10 nucleotidesequences of the present invention were obtained for both DNA strands,and are believed to be 100% accurate. However, as is known in the art,nucleotide sequence obtained by such automated methods may contain someerrors. Nucleotide sequences determined by automation are typically atleast about 90%, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of a given nucleicacid molecule. The actual sequence may be more precisely determinedusing manual sequencing methods, which are well known in the art. Anerror in sequence which results in an insertion or deletion of one ormore nucleotides may result in a frame shift in translation such thatthe predicted amino acid sequence will differ from that which would bepredicted from the actual nucleotide sequence of the nucleic acidmolecule, starting at the point of the mutation. The sel-10 DNA of thepresent invention includes cDNA, chemically synthesized DNA, DNAisolated by PCR, genomic DNA, and combinations thereof. Genomic sel-10DNA may be obtained by screening a genomic library with the sel-10 cDNAdescribed herein, using methods that are well known in the art. RNAtranscribed from sel-10 DNA is also encompassed by the presentinvention.

Due to the degeneracy of the genetic code, two DNA sequences may differand yet encode identical amino acid sequences. The present inventionthus provides isolated nucleic acid molecules having a polynucleotidesequence encoding any of the sel-10 polypeptides of the invention,wherein said polynucleotide sequence encodes a sel-10 polypeptide havingthe complete amino acid sequence of SEQ ID NOs:3-10, or fragmentsthereof.

Also provided herein are purified sel-10 polypeptides, both recombinantand non-recombinant. Variants and derivatives of native sel-10 proteinsthat retain any of the biological activities of sel-10 are also withinthe scope of the present invention. As is described above, the sel-10polypeptides of the present invention share homology with yeast CDC4. AsCDC4 is known to catalyze ubiquitination of specific cellular proteins(Feldman et al., Cell 91:221 (1997)), it may be inferred that sel-10will also have this activity. Assay procedures for demonstrating suchactivity are well known, and involve reconstitution of theubiquitinating system using purified human sel-10 protein together withthe yeast proteins Cdc4p, Cdc53p and Skp1p, or their human orthologs,and an E1 enzyme, the E2 enzyme Cdc34p or its human ortholog, ubiquitin,a target protein and an ATP regenerating system (Feldman et al., 1997).Skp1p associates with Cdc4p through a protein domain called an F-box(Bai et al., Cell 86:263 (1996)). The F-box protein motif is found inyeast CDC4, C. elegans sel-10, mouse sel-10 and human sel-10. The sel-10ubiquitination system may be reconstituted with the C. eleganscounterparts of the yeast components, e.g., cul-1 (also known as lin-19)protein substituting for Cdc53p (Kipreos et al., Cell 85:829 (1996)) andthe protein F46A9 substituting for Skp1p, or with their mammaliancounterparts, e.g., Cul-2 protein substituting for Cdc53p (Kipreos etal., 1996) and mammalian Skp1p substituting for yeast Skp1p. Aphosphorylation system provided by a protein kinase is also included inthe assay system as per Feldman et al., 1997.

Sel-10 variants may be obtained by mutation of native sel-10-encodingnucleotide sequences, for example. A sel-10 variant, as referred toherein, is a polypeptide substantially homologous to a native sel-10 butwhich has an amino acid sequence different from that of native sel-10because of one or more deletions, insertions, or substitutions in theamino acid sequence. The variant amino acid or nucleotide sequence ispreferably at least about 80% identical, more preferably at least about90% identical, and most preferably at least about 95% identical, to anative sel-10 sequence. Thus, a variant nucleotide sequence whichcontains, for example, 5 point mutations for every one hundrednucleotides, as compared to a native sel-10 gene, will be 95% identicalto the native protein. The percentage of sequence identity, also termedhomology, between a native and a variant sel-10 sequence may also bedetermined, for example, by comparing the two sequences using any of thecomputer programs commonly employed for this purpose, such as the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), whichuses the algorithm of Smith and Waterman (Adv. Appl. Math. 2: 482-489(1981)).

Alterations of the native amino acid sequence may be accomplished by anyof a number of known techniques. For example, mutations may beintroduced at particular locations by procedures well known to theskilled artisan, such as oligonucleotide-directed mutagenesis, which isdescribed by Walder et al. (Gene 42:133 (1986)); Bauer et al. (Gene37:73 (1985)); Craik (BioTechniques, January 1985, pp. 12-19); Smith etal. (Genetic Engineering: Principles and Methods, Plenum Press (1981));and U.S. Pat. Nos. 4,518,584 and 4,737,462.

Sel-10 variants within the scope of the invention may compriseconservatively substituted sequences, meaning that one or more aminoacid residues of a sel-10 polypeptide are replaced by different residuesthat do not alter the secondary and/or tertiary structure of the sel-10polypeptide. Such substitutions may include the replacement of an aminoacid by a residue having similar physicochemical properties, such assubstituting one aliphatic residue (Ile, Val, Leu or Ala) for another,or substitution between basic residues Lys and Arg, acidic residues Gluand Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, oraromatic residues Phe and Tyr. Further information regarding makingphenotypically silent amino acid exchanges may be found in Bowie et al.,Science 247:1306-1310 (1990). Other sel-10 variants which might retainsubstantially the biological activities of sel-10 are those where aminoacid substitutions have been made in areas outside functional regions ofthe protein.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringentconditions to a portion of the nucleic acid molecules described above,e.g., to at least about 15 nucleotides, preferably to at least about 20nucleotides, more preferably to at least about 30 nucleotides, and stillmore preferably to at least about from 30 to at least about 100nucleotides, of one of the previously described nucleic acid molecules.Such portions of nucleic acid molecules having the described lengthsrefer to, e.g., at least about 15 contiguous nucleotides of thereference nucleic acid molecule. By stringent hybridization conditionsis intended overnight incubation at about 42/C for about 2.5 hours in6×SSC/0.1% SDS, followed by washing of the filters in 1.0×SSC at 65/C,0.1% SDS.

Fragments of the sel-10-encoding nucleic acid molecules describedherein, as well as polynucleotides capable of hybridizing to suchnucleic acid molecules may be used as a probe or as primers in apolymerase chain reaction (PCR). Such probes may be used, e.g., todetect the presence of sel-10 nucleic acids in in vitro assays, as wellas in Southern and northern blots. Cell types expressing sel-10 may alsobe identified by the use of such probes. Such procedures are well known,and the skilled artisan will be able to choose a probe of a lengthsuitable to the particular application. For PCR, 5′ and 3′ primerscorresponding to the termini of a desired sel-10 nucleic acid moleculeare employed to isolate and amplify that sequence using conventionaltechniques.

Other useful fragments of the sel-10 nucleic acid molecules areantisense or sense oligonucleotides comprising a single-stranded nucleicacid sequence capable of binding to a target sel-10 mRNA (using a sensestrand), or sel-10 DNA (using an antisense strand) sequence.

In another aspect, the invention includes sel-10 polypeptides with orwithout associated native pattern glycosylation. Sel-10 expressed inyeast or mammalian expression systems (discussed below) may be similarto or significantly different from a native sel-10 polypeptide inmolecular weight and glycosylation pattern. Expression of sel-10 inbacterial expression systems will provide non-glycosylated sel-10.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Sel-10polypeptides may be recovered and purified from recombinant cellcultures by well-known methods, including ammonium sulfate or ethanolprecipitation, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. In a preferred embodiment, high performance liquidchromatography (HPLC) is employed for purification.

The present invention also relates to vectors comprising thepolynucleotide molecules of the invention, as well as host celltransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be joined to a vector, which generally includes aselectable marker and an origin of replication, for propagation in ahost. Because the invention also provides sel-10 polypeptides expressedfrom the polynucleotide molecules described above, vectors for theexpression of sel-10 are preferred. The vectors include DNA encoding anyof the sel-10 polypeptides described above or below, operably linked tosuitable transcriptional or translational regulatory sequences, such asthose derived from a mammalian, microbial, viral, or insect gene.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, mRNA ribosomal binding sites, and appropriatesequences which control transcription and translation. Nucleotidesequences are operably linked when the regulatory sequence functionallyrelates to the DNA encoding sel-10. Thus, a promoter nucleotide sequenceis operably linked to a sel-10 DNA sequence if the promoter nucleotidesequence directs the transcription of the sel-10 sequence.

Selection of suitable vectors to be used for the cloning ofpolynucleotide molecules encoding sel-10, or for the expression ofsel-10 polypeptides, will of course depend upon the host cell in whichthe vector will be transformed, and, where applicable, the host cellfrom which the sel-10 polypeptide is to be expressed. Suitable hostcells for expression of sel-10 polypeptides include prokaryotes, yeast,and higher eukaryotic cells, each of which is discussed below.

The sel-10 polypeptides to be expressed in such host cells may also befusion proteins which include regions from heterologous proteins. Suchregions may be included to allow, e.g., secretion, improved stability,or facilitated purification of the polypeptide. For example, a sequenceencoding an appropriate signal peptide can be incorporated intoexpression vectors. A DNA sequence for a signal peptide (secretoryleader) may be fused in-frame to the sel-10 sequence so that sel-10 istranslated as a fusion protein comprising the signal peptide. A signalpeptide that is functional in the intended host cell promotesextracellular secretion of the sel-10 polypeptide. Preferably, thesignal sequence will be cleaved from the sel-10 polypeptide uponsecretion of sel-10 from the cell. Non-limiting examples of signalsequences that can be used in practicing the invention include the yeastI-factor and the honeybee melatin leader in sf9 insect cells.

In a preferred embodiment, the sel-10 polypeptide will be a fusionprotein which includes a heterologous region used to facilitatepurification of the polypeptide. Many of the available peptides used forsuch a function allow selective binding of the fusion protein to abinding partner. For example, the sel-10 polypeptide may be modified tocomprise a peptide to form a fusion protein which specifically binds toa binding partner, or peptide tag. Non-limiting examples of such peptidetags include the 6-His tag, thioredoxin tag, FLAG tag, hemaglutinin tag,GST tag, and OmpA signal sequence tag. As will be understood by one ofskill in the art, the binding partner which recognizes and binds to thepeptide may be any molecule or compound including metal ions (e.g.,metal affinity columns), antibodies, or fragments thereof, and anyprotein or peptide which binds the peptide. These tags may be recognizedby fluorescein or rhodamine labeled antibodies that react specificallywith each type of tag.

Suitable host cells for expression of sel-10 polypeptides includeprokaryotes, yeast, and higher eukaryotic cells. Suitable prokaryotichosts to be used for the expression of sel-10 include bacteria of thegenera Escherichia, Bacillus, and Salmonella, as well as members of thegenera Pseudomonas, Streptomyces, and Staphylococcus. For expression in,e.g., E. coli, a sel-10 polypeptide may include an N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in aprokaryotic host. The N-terminal Met may optionally then be cleaved fromthe expressed sel-10 polypeptide.

Expression vectors for use in prokaryotic hosts generally comprise oneor more phenotypic selectable marker genes. Such genes generally encode,e.g., a protein that confers antibiotic resistance or that supplies anauxotrophic requirement. A wide variety of such vectors are readilyavailable from commercial sources. Examples include pSPORT vectors, pGEMvectors (Promega), pPROEX vectors (LTI, Bethesda, Md.), Bluescriptvectors (Stratagene), and pQE vectors (Qiagen).

Sel-10 may also be expressed in yeast host cells from genera includingSaccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S.cerevisiae and P. pastoris. Yeast vectors will often contain an originof replication sequence from a 2T yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene.

Vectors replicable in both yeast and E. coli (termed shuttle vectors)may also be used. In addition to the above-mentioned features of yeastvectors, a shuttle vector will also include sequences for replicationand selection in E. coli. Direct secretion of sel-10 polypeptidesexpressed in yeast hosts may be accomplished by the inclusion ofnucleotide sequence encoding the yeast I-factor leader sequence at the5′ end of the sel-10-encoding nucleotide sequence.

Insect host cell culture systems may also be used for the expression ofSel-10 polypeptides. In a preferred embodiment, the sel-10 polypeptidesof the invention are expressed using a baculovirus expression system.Further information regarding the use of baculovirus systems for theexpression of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988).

In another preferred embodiment, the sel-10 polypeptide is expressed inmammalian host cells. Non-limiting examples of suitable mammalian celllines include the COS-7 line of monkey kidney cells (Gluzman et al.,Cell 23:175 (1981)) and Chinese hamster ovary (CHO) cells.

The choice of a suitable expression vector for expression of the sel-10polypeptides of the invention will of course depend upon the specificmammalian host cell to be used, and is within the skill of the ordinaryartisan. Examples of suitable expression vectors include pcDNA3(Invitrogen) and pSVL (Pharmacia Biotech). Expression vectors for use inmammalian host cells may include transcriptional and translationalcontrol sequences derived from viral genomes. Commonly used promotersequences and enhancer sequences which may be used in the presentinvention include, but are not limited to, those derived from humancytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40(SV40). Methods for the construction of mammalian expression vectors aredisclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280(1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)); Cosman et al.(Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982.

The polypeptides of the present invention may also be used to raisepolyclonal and monoclonal antibodies, which are useful in diagnosticassays for detecting sel-10 polypeptide expression. Such antibodies maybe prepared by conventional techniques. See, for example, Antibodies: ALaboratory Manual, Harlow and Land (eds.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (1988); Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980).

The sel-10 nucleic acid molecules of the present invention are alsovaluable for chromosome identification, as they can hybridize with aspecific location on a human chromosome. There is a current need foridentifying particular sites on the chromosome, as few chromosomemarking reagents based on actual sequence data (repeat polymorphisms)are presently available for marking chromosomal location. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. The relationship between genes and diseases that havebeen mapped to the same chromosomal region can then be identifiedthrough linkage analysis, wherein the coinheritance of physicallyadjacent genes is determined. Whether a gene appearing to be related toa particular disease is in fact the cause of the disease can then bedetermined by comparing the nucleic acid sequence between affected andunaffected individuals.

The sel-10 polypeptides of the invention, and the DNA encoding them, mayalso be used to further elucidate the biological mechanism of AD, andmay ultimately lead to the identification of compounds that can be usedto alter such mechanisms. The sel-10 polypeptides of the invention are47.6% identical and 56.7% similar to C. elegans sel-10. As is describedabove, mutations to C. elegans sel-10 are known to suppress mutations tosel-12 that result in a loss-of-function for egg laying, and also tosuppress certain hypomorphic mutations to lin-12. Mutations to C.elegans sel-12 can also be rescued by either of the human AD-linkedgenes PS-1 (42.7% identical to sel-12) or PS-2 (43.4% identical tosel-12). However, human PS-1 with a familial AD-linked mutant has areduced ability to rescue sel-12 mutants (Levitan, D. et al., Proc.Natl. Acad. Sci. USA 93: 14940-14944 (1996)).

This demonstrated interchangeability of human and C. elegans genes inthe notch signaling pathway makes it reasonable to predict thatmutations of human sel-10 will suppress mutations to PS-1 or PS-2 thatlead to AD, especially in light of the predicted structure of sel-10. Asdescribed above, PS-1 and PS-2 mutations that lead to AD are those whichinterfere with the proteolytic processing of PS-1 or PS-2. The sel-10polypeptides of the invention are members of the α-transducin proteinfamily, which includes yeast CDC4, a component of an enzyme whichfunctions in the ubiquitin-dependent protein degradation pathway. Thus,human sel-10 may regulate presenilin degradation via theubiquitin-proteasome pathway. Alternatively, or in addition, humansel-10 may alter presenilin function by targeting for degradationthrough ubiquitination a modulator of presenilin activity, e.g., anegative regulator. Therefore, mutations to sel-10 may reverse thefaulty proteolytic processing of PS-1 or PS-2 which occurs as a resultof mutation to PS-1 or PS-2 or otherwise increase presenilin function.For the same reason, inhibition of sel-10 activity may also act toreverse PS-1 or PS-2 mutations. Thus, it may be hypothesized thatcompounds which inhibit either the expression or the activity of thehuman sel-10 polypeptides of the invention may reverse the effects ofmutations to PS-1 or PS-2, and thus be useful for the prevention ortreatment of AD.

Thus, C. elegans may be used as a genetic system for the identificationof agents capable of inhibiting the activity or expression of the humansel-10 polypeptides of the invention. A suitable C. elegans strain foruse in such assays lacks a gene encoding active C. elegans sel-10, andexhibits a loss-of-function for egg-laying resulting from an inactivatedsel-12 gene. Construction of C. elegans strains having aloss-of-function for egg-laying due to mutation of sel-12 may beaccomplished using routine methods, as both the sequence of sel-12(Genebank accession number U35660) and mutations to sel-12 resulting ina loss-of-function for egg laying are known (see Levitan et al., Nature377: 351-354 (1995), which describes construction of C. eleganssel-12(ar171)). An example of how to make such a strain is also given inLevitan et al. (Nature 377: 351-354 (1995)). Wild-type C. elegans sel-10in the C. elegans sel-12(ar171)), is also mutagenized using routinemethods, such as the technique used for sel-12 mutagenesis in Levitan etal., supra.

In order to identify compounds inhibiting human sel-10 activity, a DNAvector containing a human sel-10 gene encoding any of the wild-typehuman sel-10 proteins of the invention is introduced into theabove-described C. elegans strain. In a preferred embodiment, theheterologous human sel-10 gene is integrated into the C. elegans genome.The gene is then expressed, using techniques described in Levitan et al.(Proc. Natl. Acad. Sci. USA 93: 14940-14944 (1996)). Test compounds arethen administered to this strain in order to determine whether a givenagent is capable of inhibiting sel-10 activity so as to suppressmutations to sel-12 or lin-12 that result in egg-laying defects.Egg-laying in this strain is then determined, e.g. by the assaydescribed in Levitan et al. (Proc. Natl. Acad. Sci. USA 93: 14940-14944(1996)). To confirm that the compound's effect on egg-laying is due toinhibition of sel-10 activity, the action of the compound can be testedin a second biochemical or genetic pathway that is known to be affectedby loss-of-function mutations in sel-10 (e.g., further elevation oflin-12 activity in lin-12(d) hypomorphic strains). Such assays may beperformed as described in Sundarem and Greenwald (Genetics 135: 765-783(1993)).

Alternatively, compounds are tested for their ability to inhibit the E3Ubiquitin Ligating Enzyme. Assays procedures for demonstrating suchactivity are well known, and involve reconstitution of theubiquitinating system using purified human sel-10 protein together withthe yeast proteins Cdc4p, Cdc53p and Skp1p and an E1 enzyme, the E2enzyme Cdc34p, ubiquitin, a target protein and an ATP regeneratingsystem (Feldman et al., 1997). The sel-10 ubiquitination system may alsobe reconstituted with the C. elegans counterparts of the yeastcomponents, e.g., cul-1 (also known as lin-19) protein substituting forCdc53p (Kipreos et al., Cell 85:829 (1996)) and the protein F46A9substituting for Skp1p, or with their mammalian counterparts, e.g.,Cul-2 protein substituting for Cdc53p (Kipreos et al., ibid.) andmammalian Skp1p substituting for yeast Skp1p. A phosphorylation systemprovided by a protein kinase is also to be included in the assay systemas per Feldman et al., 1997.

Alternatively, cell lines which express human sel-10 due totransformation with a human sel-10 cDNA and which as a consequence haveelevated APP processing and formation of AP₁₀₋₄₀ or Aβ₁₋₄₂ may also beused for such assays as in Example 3. Compounds may be tested for theirability to reduce the elevated Aβ processing seen in the sel-10transformed cell line.

Compounds that rescue the egg-laying defect or that inhibit E3 UbiquitinLigating Enzyme are then screened for their ability to cause a reductionin the production of A-beta₁₋₄₀ or A-beta₁ ₄₂ in a human cell line. Testcompounds are used to expose IMR-32 or other human cell lines known toproduce A-beta₁₋₄₀ or A-beta₁₋₄₂ (Asami-Okada et al., Biochemistry 34:10272-10278 (1995)), or in human cell lines engineered to express humanAPP at high levels. In these assays, A-beta₁₋₄₀ or A-beta₁₋₄₂ ismeasured in cell extracts or after release into the medium by ELISA orother assays which are known in the art (Borchelt et al., Neuron 17:1005-1013 (1996); Citron et al., Nat. Med. 3: 67-72 (1997)).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Identification of a Human Homologue to C. ElegansSel-10

Results

Identification of sel-10 in ACEDB: Sel-10 maps between the clonedpolymorphisms arP3 and TCPARI just to the left of him-5 [ACEDB entrywm95p536]. Three phage lambda clones have been sequenced across theinterval, F53C11, F09F3, and F55B12. Sel-10 is reported to have homologyto yeast cdc4 [ACEDB entry wm97ab259]. Blast search revealed a singleORF with homology to yeast cdc4 (CC4_YST) within the interval defined byarP3 and TCPARI corresponding to the GenPep entry F55B12.3. F55B12.3,like yeast cdc4, is a member of the β-transducin protein family. Thisfamily is characterized by the presence of multiple WD40 repeats [Neer,E. J. et al., Nature 371: 297-300 (1994)]. Identification of a humansel-10 homologue, Incyte 028971: The GenPep entry F55B12.3 was used tosearch the LifeSeq, LifeSeq FL and EMBL data bases using tblastn. Thesearch revealed multiple homologies to β-transducin family membersincluding LIS-1 (S36113 and P43035), a gene implicated in Miller-Diekerlissencephaly, a Xenopus laevis gene, TRCPXEN (U63921), and a humancontig in LifeSeq FL, 028971. Since there also are multiple β-transducinfamily members within the C. elegans genome, these were collected usingmultiple blast searches and then clustered with the sel-10 candidategenes. Multiple alignments were performed with the DNAStar programMegalign using the Clustal method. This revealed that LIS-1 clusteredwith T03F6.F, a different β-transducin family member and thus excludedit as a candidate sel-10 homologue. TRCPXEN clustered with K10B2.1, agene which also clusters with F55B12.3 and CC4YST, while Incyte 028971clustered with sel-10. Thus, Incyte 028971 appears to encode the humanhomologue of C. elegans sel-10. Sequence homology between sel-10 and028971 is strongest in the region of the protein containing 7 repeats ofthe WD40 motif. The Incyte 028971 contig contains 44 ESTs from multiplelibraries including pancreas, lung, T-lymphocytes, fibroblasts, breast,hippocampus, cardiac muscle, colon, and others.

Public EST: Blastx searches with the DNA sequence 028971 against theTREMBLP dataset identified a single homologous mouse EST (W85144) fromthe IMAGE Library, Soares mouse embryo NbME13.5-14.5. The blastxalignment of 028971 with W85144 and then with F55B12.3 revealed a changein reading frame in 028971 which probably is due to a sequencing error.

Blastn searches of the EMBL EST database with the 028971 DNA sequencerevealed in addition to W85144, three human EST that align with thecoding sequence of 028971 and six EST that align with the 3′untranslated region of the 028971 sequence.

Protein Motifs: Two protein motifs were identified in F55B12.3 which areshared with yeast cdc4, mouse w85144 and human 028971. These are anF-box in the N-terminal domain and seven β-transducin repeats in theC-terminal domain.

Discussion

The sel-10 gene encodes a member of the β-transducin protein family.These are characterized by the presence of multiple WD40 repeats [Neer,E. J. et al., Nature 371: 297-300 (1994)]. The repeats are 20-40 aminoacids long and are bounded by gly-his (GH) and trp-asp (WD) residues.Solution of the three dimensional structure of β-transducin indicatesthat the WD40 repeats form the arms of a seven-bladed propeller likestructure [Sondek, J. et al., Nature 379: 369-74 (1996)]. Each blade isformed by four alternating pleats of beta-sheet with a pair of theconserved aspartic acid residues in the protein motif forming the limitsof one internal beta strand. WD40 repeats are found in over 27 differentproteins which represent diverse functional classes [Neer, E. J. et al.,Nature 371: 297-300 (1994)]. These regulate cellular functions includingcell division, cell fate determination, gene transcription, signaltransduction, protein degradation, mRNA modification and vesicle fusion.This diversity in function has led to the hypothesis that β-transducinfamily members provide a common scaffolding upon which multiproteincomplexes can be assembled.

The homology of sel-10, 28971 and W85144 to the yeast cdc4 gene suggestsa functional role in the ubiquitin-proteasome pathway for intracellulardegradation of protein. Mutations of the yeast cdc4 gene cause cellcycle arrest by blocking degradation of Sic1, an inhibitor of S-phasecyclin/cdk complexes [King, R. W. et al., Science 274: 1652-9 (1996)].Phosphorylation of Sic1 targets it for destruction through theubiquitin-proteasome pathway. This pathway consists of three linkedenzyme reactions that are catalyzed by multiprotein complexes[Ciechanover, A., Cell 79: 13-21 (1994); Ciechanover, A. and A. L.Schwartz, FASEB J. 8: 182-91 (1994)]. Initially, the C-terminal glycineof ubiquitin is activated by ATP to form a high energy thiol esterintermediate in a reaction catalyzed by the ubiquitin-activating enzyme,E1. Following activation, an E2 enzyme (ubiquitin conjugating enzyme)transfers ubiquitin from E1 to the protein target. In some cases, E2acts alone. In others, it acts in concert with an E3 ubiquitin-ligatingenzyme which binds the protein substrate and recruits an E2 to catalyzeubiquitination. E2 ubiquitin-conjugating enzymes comprise a fairlyconserved gene family, while E3 enzymes are divergent in sequence[Ciechanover, A., Cell 79: 13-21 (1994); Ciechanover, A. and A. L.Schwartz, FASEB J. 8: 182-91 (1994)].

In yeast, mutation of the E2 ubiquitin-conjugating enzyme, cdc34, causescell cycle arrest through failure to degrade the Sic1 inhibitor of theS-phase cyclin/cdk complex [King, R. W. et al., Science 274: 1652-9(1996)]. Sic1 normally is degraded as cells enter the G1-S phasetransition, but in the absence of cdc34, Sic1 escapes degradation andits accumulation causes cell cycle arrest. Besides cdc34, cdc4 is one ofthree other proteins required for the G1-S phase transition. The othertwo are cdc53 and Skp1. As discussed above, cdc4 contains two structuralmotifs, seven WD40 repeats (which suggests that the protein forms abeta-propeller) and a structural motif shared with cyclin F which is aninteraction domain for Skp1 [Bai, C. et al., Cell 86: 263-74 (1996)].Insect cell lysates containing cdc53, cdc4 and skp1 (and also ubiquitin,cdc34 and E1) can transfer ubiquitin to Sic1 suggesting that one or moreof these components functions as an E3 ubiquitin-ligating enzyme [King,R. W. et al., Science 274: 1652-9 (1996)]. Increased expression ofeither cdc4 or Skp1 partially rescues loss of the other.

In C. elegans, mutation of sel-10 has no visible phenotype indicatingthat sel-10 does not play a role in regulation of the cell-cycle. Aclosely related, C. elegans β-transducin family member, K10B2.6 may playthat role as it clusters with the gene TRCP_XEN from Xenopus laeviswhich rescues yeast cell cycle mutants arrested in late anaphase due toa failure to degrade cyclin B [Spevak, W. et al., Mol. Cell. Biol. 13:4953-66 (1993)]. If sel-10 does encode a component of an E3-ubiquitinligating enzyme, how might it suppress sel-12 and enhance lin-12mutations? The simplest hypothesis is that sel-10 regulates degradationof both proteins via the ubiquitin-proteasome pathway. Both sel-12 andlin-12 are transmembrane proteins. Sel-12 crosses the membrane 8 timessuch that its N- and C-termini face the cytosol [Kim, T. W. et al., J.Biol. Chem. 272: 11006-10 (1997)], while lin-12 is a type 1transmembrane protein (Greenwald, I. and G. Seydoux, Nature 346: 197-9(1990)). Both are ubiquitinated, and in the case of human PS2, steadystate levels increase in cells treated with an inhibitor of theproteasome, N-acetyl-L-leucinal-L-norleucinal and lactacystin (Li, X.and I. Greenwald, Neuron. 17: 1015-21 (1996)). Alternatively, sel-10 maytarget for degradation of a negative regulator of presenilin function.

The genetic analysis and protein function suggested by homology to cdc4implies that drug inhibitors of human sel-10 may increase steady statelevels of human presenilins. This could potentiate activity of thepresenilin pathway and provide a means for therapeutic intervention inAlzheimer's disease.

Example 2 5′ RACE Cloning of a Human cDNA Encoding Sel-10, an ExtragenicSuppressor of Presenilin Mutations in C. elegans

Materials and Methods

Oligonucleotide primers for the amplification of the sel-10 codingsequence from C. elegans cDNA were prepared based on the sequence ofF55B12.3, identified in Example 1 as the coding sequence for C. eleganssel-10. The primers prepared were:5′-CGGGATCCACCATGGATGATGGATCGATGACACC-3′ (SEQ ID NO:11) and5′-GGAATTCCTTAAGGGTATACAGCATCAAAGTCG-3′ (SEQ ID NO:12). C. elegans mRNAwas converted to cDNA using a BRL Superscript II Preamplification kit.The PCR product was digested with restriction enzymes BamHI and EcoRI(LTI, Gaithersberg, Md.) and cloned into pcDNA3.1 (Invitrogen). Twoisolates were sequenced (ABI, Perkin-Elmer Corp).

The sequence of Incyte clone 028971 (encoding a portion of the humanhomologue of C. elegans sel-10), was used to design four antisenseoligonucleotide primers: 5′-TCACTTCATGTCCACATCAAAGTCC-3′ (SEQ ID NO:13),5′-GGTAATTACAAGTTCTTGTTGAACTG (SEQ ID NO:14),5′-CCCTGCAACGTGTGTAGACAGG-3′ (SEQ ID NO:15), and5′-CCAGTCTCTGCATTCCACACTTTG-3′ (SEQ ID NO:16) to amplify the missing 5′end of human sel-10. The Incyte LifeSeq “Electronic Northern” analysiswas used to identify tissues in which sel-10 was expressed. Two ofthese, hippocampus and mammary gland, were chosen for 5′ RACE cloningusing a CloneTech Marathon kit and prepared Marathon-ready cDNA fromhippocampus and mammary gland. PCR products were cloned into the TAvector pCR3.1 (Invitrogen), and isolates were sequenced. An alternate 5′oligonucleotide primer was also designed based on Incyte clones whichhave 5′ ends that differ from the hippocampal sel-10 sequence:5′-CTCAGACAGGTCAGGACATTTGG-3′. (SEQ ID NO:17)

Blastn was used to search Incyte databases LifeSeq and LifeSeqFL. Gapalignments and translations were performed with GCG programs (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, Madison Wis.).

Results

The coding sequence of the C. elegans sel-10: The predicted codingsequence of the C. elegans sel-10, F55B12.3, had originally beendetermined at the Genome Sequencing Center, Washington University, St.Louis, by using the computer program GeneFinder to predict introns andexons in the genomic cosmid F55B 12. The hypothetical cDNA sequence wasconfirmed by amplifying this region from C. elegans cDNA, cloning, andsequencing it.

The coding sequence of the human sel-10 gene homologue: All of the028971 antisense oligonucleotides amplified a 5′ RACE product from humanhippocampal and mammary cDNA. The longest PCR product from thehippocampal reactions was cloned and sequenced. This PCR reaction wasdesigned to generate products which end at the predicted stop codon. Twoisolates contained identical sequence which begins 880 bases before thebeginning of the 028971 sequence. This sequence was confirmed bycomparison with spanning Incyte cDNA clones. The Incyte clones thatspanned the 5′ end of the human sel-10 homologue were not annotated asF55B12.3, as the homology in this region between the human and C.elegans genes is low, and as the overlap between these clones and theannotated clones happened to be too small for them to be clustered inthe Incyte database or uncovered by our blasting the Incyte databasewith the 028971 sequence.

The predicted protein sequences of human sel-10 have 47.6% identity and56.7% similarity to C. elegans sel-10. The N-terminus of the humansel-10 sequence begins with 4 in-frame methionines. In addition to theWD40 repeats described above, the human sequence also contains a regionhomologous to the CDC4 F-box for binding Skp1, as expected for a sel-10homologue.

Different Human Sel-10 mRNAs Expressed in Mammary and HippocampalTissues:

Several additional human sel-10 ESTs which differ from the hippocampalsequence were identified. These are an exact match, which indicates thatthe alternative transcript is probably real. Comparison of thesesequences with the human hippocampal sel-10 sequence shows divergenceprior to the 4th in-frame methionine and then exact sequence matchthereafter. An oligonucleotide primer specific for the 5′ end of thisalternative transcript was found to amplify a product from mammary butnot hippocampal cDNA. This indicates either that the human sel-10transcript undergoes differential splicing in a tissue-specific fashionor that the gene contains multiple, tissue specific promoters.

Discussion

5′RACE and PCR amplification were used to clone a full-length cDNAencoding the human homologue of the C. elegans gene, sel-10. Sequenceanalysis confirms the earlier prediction that sel-10 is a member of theCDC4 family of proteins containing F-Box and WD40 Repeat domains. Twovariants of the human sel-10 cDNA were cloned from hippocampus andmammary gland which differed in 5′ sequence preceding the apparent siteof translation initiation. This implies that the gene may have two ormore start sites for transcription initiation which are tissue-specificor that the pattern of exon splicing is tissue-specific.

EXAMPLE 3 Expression Of Epitope-Tagged Sel-10 In Human Cells, andPerturbation Of Amyloid β Peptide Processing By Human Sel-10

Materials And Methods

Construction of Epitope-Tagged Sel-10: Subcloning, Cell Growth andTransfection:

An EcoR1 site was introduced in-frame into the human sel-10 cDNA using apolymerase chain reaction (PCR) primed with the oligonucleotides 237(5′-GGAATTCCATGAAAAGATTGGACCATGGTTCTG-3′) (SEQ ID NO:18) and 206(5′-GGAATTCCTCACTTCATGT-CACATCAAAGTCCAG-3′) (SEQ ID NO:19). Theresulting PCR product was cloned into the EcoRI site of the vectorpCS2+MT. This fused a 5′ 6-myc epitope tag in-frame to the fifthmethionine of the hippocampal sel-10 cDNA, i.e., upstream of nucleotide306 of the sequence given in SEQ ID NO:1. The nucleotide sequence ofthis construct, designated 6myc-N-sel-10, is given in SEQ ID NO: 20,while the amino acid sequence of the polypeptide encoded thereby isgiven in SEQ ID NO: 21. The hippocampal and mammary sel-10 cDNA divergeupstream of this methionine. A PS1 cDNA with a 3′-FLAG tag (PS1—C-FLAG)was subcloned into the pcDNA3.1 vector. An APP cDNA containing theSwedish NL mutation and an attenuated ER retention sequence consistingof the addition of a di-lysyl motif to the C-terminus of APP695(APP695NL-KK) was cloned into vector pIRES-EGFP (Mullan et al., NatGenet 1992 August;1(5):345-7). HEK293 and IMR32 cells were grown to 80%confluence in DMEM with 10% FBS and transfected with the above cDNA. Atotal of 10 mg total DNA/6×10⁶ cells was used for transfection with asingle plasmid. For cotransfections of multiple plasmids, an equalamount of each plasmid was used for a total of 10 mg DNA usingLipofectAmine (BRL).

In order to construct C-term V5 his tagged sel-10 and the C-term mychistagged sel-10, the coding sequence of human hippocampal sel-10 wasamplified using oligonucleotides primers containing a KpnI restrictionsite on the 5′ primer: 5′-GGGTACCCCTCATTATTCCCTCGAGTTCTTC-3′ (SEQ IDNO:22) and an EcoRI site on the 3′ primer:5′-GGAATTCCTTCATGTCCACATCAAAGTCC-3′ (SEQ ID NO:23), using the originalhuman sel-10 RACE pcr product as template. The product was digested withboth KpnI and EcoRI and cloned into either the vector pcDNA6/V5-His A orpcDNA3.1/Myc-His(+) A (Invitrogen). The nucleotide sequence ofindependent isolates was confirmed by dideoxy sequencing. The nucleotidesequence of the C-term V5 his tagged sel-10 is given in SEQ ID NO: 24,while the amino acid sequence of the polypeptide encoded thereby isgiven in SEQ ID NO: 25. The nucleotide sequence of independent isolateswas confirmed by dideoxy sequencing. The nucleotide sequence of theC-term mychis tagged sel-10 is given in SEQ ID NO: 26, while the aminoacid sequence of the polypeptide encoded thereby is given in SEQ ID NO:27.

Clonal Selection of transformed cells by FACS: Cell samples wereanalyzed on an EPICS Elite ESP flow cytometer (Coulter, Hialeah, Fla.)equipped with a 488 nm excitation line supplied by an air-cooled argonlaser. EGFP emission was measured through a 525 nm band-pass filter andfluorescence intensity was displayed on a 4-decade log scale aftergating on viable cells as determined by forward and right angle lightscatter. Single green cells were separated into each well of one 96 wellplate containing growth 10 medium without G418. After a four dayrecovery period, G418 was added to the medium to a final concentrationof 400 mg/ml. Wells with clones were expanded from the 96 well plate toa 24 well plate and then to a 6 well plate with the fastest growingcolonies chosen for expansion at each passage.

Immunofluorescence: Cells grown on slides were fixed 48 hrs aftertransfection with 4% formaldehyde and 0.1% Triton X-100 in PBS for 30min on ice and blocked with 10% Goat serum in PBS (blocking solution) 1hr RT (i.e., 25° C.), followed by incubation with mouse anti-myc (10mg/ml) or rabbit anti-FLAG (0.5 mg/ml) antibody 4° C. O/N and thenfluorescein-labeled goat anti-mouse or anti-rabbit antibody (5 mg/ml) inblocking solution 1 hr at 25° C.

Western blotting: Cell lysates were made 48 hrs after transfection byincubating 10⁵ cells with 100 ml TENT (50 mM Tris-HCl pH 8.0, 2 mM EDTA,150 mM NaCl, 1% Triton X-100, 1× protease inhibitor cocktail) 10 min onice followed by centrifugation at 14,000 g. The supernatant was loadedon 4-12% NuPage gels (50 mg protein/lane) and electrophoresis andtransfer were conducted using an Xcell II Mini-Cell system (Novex). Theblot was blocked with 5% milk in PBS 1 hr RT and incubated with anti-mycor anti-FLAG antibody (described in “Immunofluorescence” above) 4° C.O/N, then sheep anti-mouse or anti-rabbit antibody-HRP (0.1 mg/ml) 1 hrRT, followed by Supersignal (Pierce) detection.

ELISA: Cell culture supernatant or cell lysates (100 ml formic acid/10⁶cells) were assayed in the following double antibody sandwich ELISA,which is capable of detecting levels of Aβ₁₋₄₀ and Aβ₁₋₄₂ peptide inculture supernatant.

Human Aβ1-40 or 1-42 was measured using monoclonal antibody (mAb) 6E10(Senetek, St. Louis, Mo.) and biotinylated rabbit antiserum 162 or 164(NYS Institute for Basic Research, Staten Island, N.Y.) in a doubleantibody sandwich ELISA. The capture antibody 6E10 is specific to anepitope present on the N-terminal amino acid residues 1-16 of hAβ. Theconjugated detecting antibodies 162 and 164 are specific for hAp 1-40and 1-42, respectively. The sandwich ELISA was performed according tothe method of Pirttila et al. (Neurobiology of Aging 18: 121-7 (1997)).Briefly, a Nunc Maxisorp 96 well immunoplate was coated with 100 μl/wellof mAb 6E10 (5 μg/ml) diluted in 0.1M carbonate-bicarbonate buffer, pH9.6 and incubated at 4° C. overnight. After washing the plate 3× with0.01M DPBS (Modified Dulbecco's Phosphate Buffered Saline (0.008M sodiumphosphate, 0.002M potassium phosphate, 0.14M sodium chloride, 0.01 Mpotassium chloride, pH 7.4) from Pierce, Rockford, Ill.) containing0.05% of Tween-20 (DPBST), the plate was blocked for 60 min with 200 μlof 10% normal sheep serum (Sigma) in 0.01M DPBS to avoid non-specificbinding. Human Aβ₁₋₄₀ or 1-42 standards 100 μl/well (Bachem, Torrance,Calif.) diluted, from a 1 mg/ml stock solution in DMSO, in nontransfected conditioned cell medium was added after washing the plate,as well as 100 μl/well of sample i.e. filtered conditioned medium oftransfected cells. The plate was incubated for 2 hours at roomtemperature and 4° C. overnight. The next day, after washing the plate,100 μl/well biotinylated rabbit antiserum 162 1:400 or 164 1:50 dilutedin DPBST+0.5% BSA was added and incubated at room temperature for 1 hr15 min. Following washes, 100%1/well neutravidin-horseradish peroxidase(Pierce, Rockford, II) diluted 1:10,000 in DPBST was applied andincubated for 1 hr at room temperature. After the last washes 100μl/well of o-phenylnediamine dihydrochloride (Sigma Chemicals, St.Louis, Mo.) in 50 mM citric acid/100 mM sodium phosphate buffer (SigmaChemicals, St. Louis, Mo.), pH 5.0, was added as substrate and the colordevelopment was monitored at 450 nm in a kinetic microplate reader for20 min. using Soft max Pro software.

Results

Transfection of HEK293 cells: Transfection efficiency was monitoredthrough the use of vectors that express green fluorescent protein (GFP)or by immunofluorescent detection of epitope-tagged sel-10 or PS1. AnN-terminal 6-myc epitope was used to tag human sel-10 (6myc-N-sel-10),while PS1 was tagged with a C-terminal FLAG epitope (PS1-C-FLAG). APP695was modified by inclusion of the Swedish NL mutation to increase Aβprocessing and an attenuated endoplasmic reticulum (ER) retention signalconsisting of a C-terminal di-lysine motif (APP695NL-KK). The di-lysinemotif increases Aβ processing about two fold. The APP695NL-KK constructwas inserted into the first cistron of a bicistronic vector containingGFP (pIRES-EGFP, Invitrogen) to allow us to monitor transfectionefficiency. Transfection efficiency in HEK293 cells was about 50% fortransfections with a single plasmid DNA. For cotransfections with twoplasmids, about 30-40% of the cells expressed both proteins as detectedby double immunofluorescence.

Expression of recombinant protein in transfected HEK293 cells wasconfirmed by Western blot as illustrated for PS 1-C-FLAG and6myc-N-sel-10 (FIG. 1A). In the case of cotransfections with threeplasmids (PS1—C-FLAG+6myc-N-sel-10+APP), all three proteins weredetected in the same cell lysate by Western blot (FIG. 1B) usingappropriate antibodies.

Effect of 6myc-N-sel-10 and PS1-C-FLAG on Aβ processing: Cotransfectionof APP695NL-KK with 6myc-N-sel-10 or PS1—C-FLAG into HEK293 cellsincreased the release of Ab 1-40 and Ab 1-42 peptide into the culturesupernatant by 2- to 3-fold over transfections with just APP695NL-KK(Table 1). Cotransfection of APP695NL-KK with both 6myc-N-sel-10 and PS1-C-FLAG increased Ab release still further (i.e., 4- to 6-foldincrease). In contrast, the ratio of Ab1-42/(Ab1-40+Ab1-42) releasedinto the supernatant decreased about 50%. The subtle decrease in theratio of Ab 1-42/(Ab 1-40+Ab 1-42) reflects the larger increase in Ab1-40 relative to Ab 1-42. Neither 6myc-N-sel-10 nor PS 1-C-FLAG affectedendogenous Ab production in HEK293 cells. Similar observations were alsoobtained in IMR32 cells (Table 2). However, IMR32 cells transfected lesswell than HEK293 cells, so the stimulation of APP695NL-KK processing bycotransfection with 6myc-N-sel-10 or PS 1-C-FLAG was lower.

Levels of Ab 1-40 expressed in HEK293 cells transfected with APP695NL-KKwere sufficient to measure Ab peptide in both the culture supernatantand cell pellet. Considerably more Ab 1-40 is detected in the HEK293cell pellet than in the supernatant in cells transfected with justAPP695NL-KK. Cotransfection with 6myc-N-sel-10 or PS1—C-FLAGproportionately decreased Ab 1-40 in the cell pellet and increased Ab inthe culture supernatant. This implies that 6myc-N-sel-10 and PS1—C-FLAGalter processing or trafficking of APP such that proportionately more Abis released from the cell.

Effect of 6myc-N-sel-10 and PS1—C-FLAG expression on endogenous Aβprocessing: The effect of 6myc-N-sel-10 on the processing of endogenousAPP by human cells was assessed by creating stably transformed HEK293cell lines expressing these proteins. Two cell lines expressing6myc-N-sel-10 were derived (sel-10/2 & sel-10/6) as well as a controlcell line transformed with pcDNA3.1 vector DNA. Both 6myc-N-sel-10 celllines expressed the protein as shown by Western blot analysis.Endogenous production of Ab 1-40 was increased in both 6myc-N-sel-10cell lines in contrast to vector DNA transformed cells Table 3). Inaddition, stable expression of 6myc-N-sel-10 significantly increased Abproduction after transfection with APP695NL-KK plasmid DNA (Table 3).Similar results were obtained with 6 stable cell lines expressing PS1-C-FLAG. All 6 cell lines showed significant elevation of endogenous Aβprocessing and all also showed enhanced processing of Ab aftertransfection with APP695NL-KK (Table 3). In addition, the increase of Aβprocessing seen with 6myc-N-sel-10 was also seen with sel-10 tagged atthe C-terminus with either mychis or v5his (See Table 4). BothC-terminal and N-terminal tags resulted in an increase in Aβ processing.

Discussion

These data suggest that, when over expressed, 6myc-N-sel-10 as well asPS 1-C-FLAG alter Aβ processing in both transient and stable expressionsystems. A 6-myc epitope tag was used in these experiments to allowdetection of sel-10 protein expression by Western blot analysis. If asits sequence homology to yeast CDC4 suggests, sel-10 is an E2-E3ubiquitin ligase, it should be possible to identify the proteins ittargets for ubiquitination. Since the presenilins are degraded via theubiquitin-proteasome pathway, PS1 & PS2 are logical targets of sel-10catalyzed ubiquitination [Kim et al., J. Biol. Chem. 272:11006-11010(1997)]. How sel-10 affects Aβ processing is not understood at thispoint. In the future, it will be necessary to determine if sel-10 & PS1increase Aβ processing by altering production, processing, transport, orturn-over of APP, and whether the effect of PS1 is mediated or regulatedby sel-10.

These experiments suggest that sel-10 is a potential drug target fordecreasing Ab levels in the treatment of AD. They also show that C.elegans is an excellent model system in which to investigate presenilinbiology in the context of AD. Thus, as is shown by cotransfectionexperiments, as well as in stable transformants, expression of6myc-N-sel10 or PS1-C-FLAG increases Aβ processing. An increase in Aβprocessing was seen in both HEK293 cells and IMR32 cells aftercotransfection of 6myc-N-sel 10 or PS 1-C-FLAG with APP695NL-KK. Instable transformants of HEK293 cells expressing 6myc-Sel10 orPS1—C-FLAG, an increase in endogenous Aβ processing was observed, aswell as an increase in Aβ processing after transfection withAPP695NL-KK. This suggests that inhibitors of either sel-10 and/or PS1,may decrease Aβ processing, and could have therappeutic potential forAlzheimer's disease.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the invention.

The entire disclosure of all publications cited herein are herebyincorporated by reference. TABLE 1 Effect of 6myc-N-sel-10 andPS1-C-FLAG transient transfection on Ab levels in HEK293 cellsupernatants. Plasmids Ab1-42/total Ab Transfected Ab1-42 ng/ml Ab1-40ng/ml ng/ml pcDNA3 81 ± 20 231 ± 50  0.26 ± 0.05 6myc-N-sel-10 67 ± 7 246 ± 34  0.21 ± 0.03 PS1-C-FLAG 75 ± 18 227 ± 45  0.25 ± 0.03PS1-C-FLAG + 77 ± 21 220 ± 26  0.25 ± 0.03 6myc-N-sel-10 APP695NL-KK 141± 27  896 ± 103 0.14 ± 0.02 APP695NL-KK + 308 ± 17  2576 ± 190  0.11 ±0.00 6myc-N-sel-10 APP695NL-KK + 364 ± 39  3334 ± 337  0.09 ± 0.00PS1-C-FLAG APP695NL-KK + 550 ± 20  5897 ± 388  0.09 ± 0.00 PS1-C-FLAG +6myc-N-sel-10

TABLE 2 Effect of 6myc-N-sel-10 and PS1-C-FLAG transient transfection onAb levels in IMR32 cell supernatants. Plasmids Ab1-42/total AbTransfected Ab1-42 ng/ml Ab1-40 ng/ml ng/ml pcDNA3 65 ± 3  319 ± 1460.19 ± 0.06 6myc-N-sel-10 63 ± 0  246 ± 53  0.21 ± 0.04 PS1-C-FLAG 67 ±6  307 ± 79  0.18 ± 0.04 PS1-C-FLAG + 67 ± 6  302 ± 94  0.20 ± 0.086myc-N-sel-10 APP695NL-KK 66 ± 5  348 ± 110 0.17 ± 0.05 APP695NL-KK + 75± 18 448 ± 141 0.15 ± 0.03 6myc-N-sel-10 APP695NL-KK + 63 ± 26 466 ± 72 0.12 ± 0.02 PS1-C-FLAG APP695NL-KK + 81 ± 26 565 ± 179 0.12 ± 0.01PS1-C-FLAG + 6myc-N-sel-10

TABLE 3 Endogenous and exogenous Ab1-40 and Ab1-42 levels insupernatants from stable transformants of HEK293 cells. GFP TransfectionAPP695NL-KK Transfection Ab1-40 Ab1-42 Ab1-42 Stable Line ng/ml ng/mlAb1-40 ng/ml ng/ml 6myc-N-sel10/2 297 ± 29  109 ± 17  4877 ± 547  750 ±32  6myc-N-sel10/6 168 ± 18  85 ± 11 8310 ± 308  1391 ± 19  PS1-C-FLAG/297 ± 6  68 ± 8  3348 ± 68  493 ± 21  PS1-C-FLAG/8 118 ± 11  85 ± 17 3516± 364  515 ± 36  PS1-C-FLAG/9 83 ± 20 67 ± 16 2369 ± 73  350 ± 12 PS1-C-FLAG/11 152 ± 17  68 ± 13 4771 ± 325  599 ± 25  PS1-C-FLAG/12 141± 12  50 ± 10 4095 ± 210  449 ± 21  PS1-C-FLAG/13 270 ± 139 61 ± 28 6983± 304  745 ± 41  pcDNA3/1 43 ± 13 75 ± 15 1960 ± 234  61 ± 6 

TABLE 4 Sel-10 constructs with epitope tags at the N or C terminusincrease Aβ 1-40 and Aβ 1-42. construct Aβ 1-40 % increase P-value Aβ1-42 % increase P-value pcDNA 4240 ± 102  614 ± 10 6myc-N-sel-10 7631 ±465 80% 3.7 × 10⁻⁶ 1136 ± 73 46% 7.9 × 10⁻⁶ sel-10-C-mychis 5485 ± 32929% 1.8 × 10⁻⁴  795 ± 50 29% 4.0 × 10⁻⁴ sel-10-C-V5his 6210 ± 498 46%1.2 × 10⁻⁴  906 ± 73 48% 2.1 × 10⁻⁴

1. An isolated nucleic acid molecule comprising a polynucleotide havinga sequence at least 95% identical to a sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding a human sel-10polypeptide having the complete amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,and SEQ ID NO:7, or as encoded by the cDNA clone contained in ATCCDeposit No.98978; (b) a nucleotide sequence encoding a human sel-10polypeptide having the complete amino acid sequence selected from thegroup consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or asencoded by the cDNA clone contained in ATCC Deposit No. 98979; and (c) anucleotide sequence complementary to the nucleotide sequence of (a) or(b).
 2. An isolated nucleic acid molecule comprising polynucleotidewhich hybridizes under stringent conditions to a polynucleotide havingthe nucleotide sequence in (a), (b), or (c) of claim
 1. 3. The nucleicacid molecule of claim 1, wherein said polynucleotide of 1 (a) encodes ahuman sel-10 polypeptide having the complete amino acid sequence of SEQID NO:3.
 4. The nucleic acid molecule of claim 3, wherein saidpolynucleotide molecule of 1(a) comprises the nucleotide sequence ofresidues 45-1928 of SEQ ID NO:1.
 5. The nucleic acid molecule of claim1, wherein said polynucleotide of 1(a) encodes a human sel-10polypeptide having the complete amino acid sequence of SEQ ID NO:4. 6.The nucleic acid molecule of claim 5, wherein said polynucleotidemolecule of 1 (a) comprises the nucleotide sequence of residues 150-1928of SEQ ID NO:1.
 7. The nucleic acid molecule of claim 1, wherein saidpolynucleotide of 1(a) encodes a human sel-10 polypeptide having thecomplete amino acid sequence of SEQ ID NO:5.
 8. The nucleic acidmolecule of claim 7, wherein said polynucleotide molecule of 1(a)comprises the nucleotide sequence of residues 267-1928 of SEQ ID NO:1.9. The nucleic acid molecule of claim 1, wherein said polynucleotide of1(a) encodes a human sel-10 polypeptide having the complete amino acidsequence of SEQ ID NO:6.
 10. The nucleic acid molecule of claim 9,wherein said polynucleotide molecule of 1(a) comprises the nucleotidesequence of residues 291-1928 of SEQ ID NO:1.
 11. The nucleic acidmolecule of claim 1, wherein said polynucleotide of 1(a) encodes a humansel-10 polypeptide having the complete amino acid sequence of SEQ IDNO:7.
 12. The nucleic acid molecule of claim 11, wherein saidpolynucleotide molecule of 1(a) comprises the nucleotide sequence ofresidues 306-1928 of SEQ ID NO:1.
 13. The nucleic acid molecule of claim1, wherein said polynucleotide of 1(b) encodes a human sel-10polypeptide having the complete amino acid sequence of SEQ ID NO:8. 14.The nucleic acid molecule of claim 13 wherein said polynucleotidemolecule of 1(b) comprises the nucleotide sequence of residues 180-1949of SEQ ID NO:2.
 15. The nucleic acid molecule of claim 1, wherein saidpolynucleotide of 1(b) encodes a human sel-10 polypeptide having thecomplete amino acid sequence of SEQ ID NO:9.
 16. The nucleic acidmolecule of claim 15, wherein said polynucleotide molecule of 1(b)comprises the nucleotide sequence of residues 270-1949 of SEQ ID NO:2.17. The nucleic acid molecule of claim 1, wherein said polynucleotide of1(b) encodes a human sel-10 polypeptide having the complete amino acidsequence of SEQ ID NO:10.
 18. The nucleic acid molecule of claim 17,wherein said polynucleotide molecule of 1(b) comprises the nucleotidesequence of residues 327-1949 of SEQ ID NO:2.
 19. A vector comprisingthe nucleic acid molecule of claim
 1. 20. The vector of claim 19,wherein said nucleic acid molecule of claim 1 is operably linked to apromoter for the expression of a sel-10 polypeptide.
 21. A host cellcomprising the vector of claim
 19. 22. The host cell of claim 21,wherein said host is a eukaryotic host.
 23. A method of obtaining asel-10 polypeptide comprising culturing the host cell of claim 22 andisolating said sel-10 polypeptide.
 24. An isolated sel-10 polypeptidecomprising (a) an amino acid sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7,or as encoded by the cDNA clone contained in ATCC Deposit No. 98978; (b)an amino acid sequence selected from the group consisting of SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10, or as encoded by the cDNA clonecontained in ATCC Deposit No.
 98979. 25. The isolated sel-10 polypeptideof claim 24, wherein said polypeptide comprises the amino acid sequenceof SEQ ID NO:3.
 26. The isolated sel-10 polypeptide of claim 24, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:4. 27.The isolated sel-10 polypeptide of claim 24, wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:5.
 28. The isolatedsel-10 polypeptide of claim 24, wherein said polypeptide comprises theamino acid sequence of SEQ ID NO:6.
 29. The isolated sel-10 polypeptideof claim 24, wherein said polypeptide comprises the amino acid sequenceof SEQ ID NO:7.
 30. The isolated sel-10 polypeptide of claim 24, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:8. 31.The isolated sel-10 polypeptide of claim 24, wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:9.
 32. The isolatedsel-10 polypeptide of claim 24, wherein said polypeptide comprises theamino acid sequence of SEQ ID NO:10.
 33. An isolated antibody that bindsspecifically to the sel-10 polypeptide of claim
 24. 34. A cell linehaving altered Aβ processing that expresses any of the sel-10 isolatednucleic acid molecules of claim
 1. 35. The cell line of claim 34,wherein said Aβ processing is increased.
 36. The cell line of claim 34,wherein said Aβ processing is decreased.
 37. The cell line of claim 34,wherein said cell line is 6myc-N-sel10/2.
 38. The cell line of claim 34,wherein said cell line is 6myc-N-sel10/6.
 39. A method for theidentification of an agent capable of altering the ratio ofAβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ produced in any of the cell lines of claims 34, 37,and 38, comprising the steps of: (a) obtaining a test culture and acontrol culture of said cell line; (b) contacting said test culture witha test agent; (c) measuring the levels of Aβ₁₋₄₀ and Aβ₁₋₄₂ produced bysaid test culture of step (b) and said control culture; (d) calculatingthe ratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ for said test culture and said controlculture from the levels of Aβ₁₋₄₀ and Aβ₁₋₄₂ measured in step (c); and(e) comparing the ratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ measured for said testculture and said control culture in step (d); whereby a determinationthat the ratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ for said test culture is higheror lower than ratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ for said control cultureindicates that said test agent has altered the ratio ofAβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂.
 40. The method of claim 39, wherein said ratio ofAβ₁40/Aβ₁₋₄₀+Aβ₁₋₄₂ is increased by said test agent.
 41. The method ofclaim 39, wherein said ratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ is decreased bysaid test agent.